PubMed · 8757122
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M F Tuite. 1996-08-15. RNA processing. Death by decapitation for mRNA.. https://doi.org/10.1038/382577a0
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Poly-A tail length dynamics have been extensively studied from yeast to human, mostly using reporter transcripts. Recent studies have been carried out genome-wide to determine the status of poly-A tails at steady state. However, poly-A tail measurement at equilibrium gives an overall length that reflects a mixture of the different poly-A tail sizes for a single transcript. New genome-scale techniques are emerging to estimate dynamic of poly-A tails lengths, but they are not yet routine and individual validation experiments are useful. In this chapter we describe a protocol for visualizing poly-A tail lengths following transcription inhibition for a reporter mRNA using denaturing poly-acrylamide gel electrophoresis and northern blot assay. This protocol is quick to set up, requires the purchase of only a few specific reagents, does not rely on radioactivity for RNA monitoring, and can be easily implemented in any molecular biology laboratory.
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Poly(A)-binding protein, the most abundant eukaryotic mRNP protein, is known primarily for its association with polyadenylate tails of mRNA. In the yeast, Saccharomyces cerevisiae, this protein (Pabp) was found to be essential for viability and has been implicated in models featuring roles in mRNA stability and as an enhancer of translation initiation. Although the mechanism of action is unknown, it is thought to require an activity to bind poly(A) tails and an additional capacity for an interaction with 60 S ribosomal subunits, perhaps via ribosomal protein L46 (Rpl46). We have found that a significant amount of Pabp in wild-type cells is not associated with polyribosome complexes. The remaining majority, which is found in these complexes, maintains its association even in yeast cells deficient in polyadenylated mRNA and/or Rpl46. These observations suggest that Pabp may not require interaction with poly(A) tails during translation. Further treatment of polyribosome lysates with agents known to differentially disrupt components of polyribosomes indicated that Pabp may require contact with some RNA component of the polyribosome, which could be either non-poly(A)-rich sequences of the translated mRNA or possibly a component of the ribosome. These findings suggest that Pabp may possess the ability to bind to ribosomes independently of its interaction with poly(A). We discuss these conclusions with respect to current models suggesting a multifunctional binding capacity of Pabp.